Preparation of denaturing 6% polyacrylamide gels for microsatellite analysis

Preparation of denaturing 6% polyacrylamide gels for microsatellite analysis

(also for SSAP, high-resolution IRAP/ISSR and other analyses)

Saadiah Jamli and Pat Heslop-Harrison November 2003

University of Leicester

Preparation of the plates

 

 

 

 

a)      Wear gloves. Clean the plate with laboratory detergent (type used for cleaning radioactivity � e.g. 25% Lipsol) and warm water. Rinse.

b)     Clean the upper surface with 100% ethanol. Using blue rolls of tissues, polish until it ‘squeaks’.

c)      Apply a few drops of Repel-silane to the upper surface of the plate and spread evenly using blue roll.

d)     Wipe with warm water and then 100% ethanol, and let air dry.

 

Change gloves between working with Repel-silane and Bind-silane

 

a)      Clean the plate with detergent (25% Lipsol) and warm water. (You may also need to use a razor blade to remove old bits of gels that have stuck). Rinse.

b)     Clean the upper surface with 100% ethanol. Using blue roll, polish until it squeaks.

c)      Apply 20 m l Bind-silane to the upper surface of the plate and spread evenly using blue roll.

d)     Wipe with warm water and then 100% ethanol, and let air dry.

 

 

 

4.5 ml acrylamide solution (care � neurotoxin)

3.0 ml 10X TBE

14.4 g urea

Top up with deionized water to 30 ml for 20cm x 20cm plates.

Marker ladder: 5 m l hyperladder + 3 m l formamide loading buffer

PCR reaction: 3 m l PCR product + 3.0 m l formamide loading buffer

a)      Fixer (10% acetic acid)

Add 50 ml glacial acetic acid to 450 ml distilled water

b)      Silver stain

Add 3 ml 1N silver nitrate solution in 500 ml distilled water.

Then add 0.75ml formamide

c)      Developer

Dissolve 15 g sodium carbonate in 500 ml distilled water and put at 4^O C

1.      Remove the gel and separate the plates carefully with a single-edged razor blade.

2.      Place the gel in tray with the fixer and leave shaking in a fume hood for 30 minutes. Pour off fixer (save).

3.      Wash with water for 10 � 15 minutes, on shaker. Rinse again and pour off the water.

4.      Add silver-stain and leave shaking for 30 minutes (Silver stain can be re-used up to 10 times).

5.        Immediately before developing the gel, add in 75 m l sodium thiosulphate solution (0.1N) and 0.75 ml formamide to the pre-chilled developer solution.

6.      After silver-stain (step 4), agitate the gel several times for 10 seconds.

7.      Immediately agitate the gel in the developer until band development progress sufficiently.

8.      Stop the reaction by adding the fixer saved earlier (step 2). Agitate until bubbles cease.

9.      Rinse gel in water for 20 minutes.

10.  Leave to surface dry by standing vertically for 10 min.

11.  Scan the gel with a computer flatbed scanner, or photograph.